This application is a national stage filing under 35 U.S.C. xc2xa7371 of International Application No. PCT/FR97/01308.
The invention relates to a method of preserving infectious recombinant viruses, an aqueous viral suspension, and its use as medicament.
Live viruses are used for a variety of purposes, in particular as vaccines. They are particles which have a genome in the form of DNA or RNA containing the information which is useful for their replication, but which need to infect a host cell to synthesize proteins which they require.
Moreover, the possibility of integrating foreign genetic material into a viral genome has allowed so-called recombinant viruses to be generated which carry a gene of therapeutic interest and which are used to transfer this gene into specific cells of deficient patients. This is the principle of gene therapy.
The possibility of treating human diseases by gene therapy has passed from the stage of theoretical considerations to the stage of clinical applications within a few years. To transfer and express the therapeutic gene in the cells to be treated, the vast majority of the protocols described to date make use of viral vectors.
Due to the simplicity of their genome, retroviral vectors are currently amongst the most frequently used, even though they have a somewhat limited cloning capacity.
Adenoviruses, in turn, have several advantages which make them the vectors of choice for a wide range of applications. In effect, they infect many types of cells, are non-integrating, have low pathogenicity and can replicate in dividing or quiescent cells. By way of indication, their genome is composed of a linear double-stranded DNA molecule of approx. 36 kb which carries more than approx. thirty genes which are at the same time early genes required for viral replication (E1 to E4; E for early) and late structural genes (L1-L5; L for late).
Recombinant adenoviral vectors used for gene therapy purposes are deficient for replication to avoid them spreading in the environment and in the host organism. Generally, they lack most of the E1 region, and some lack the inflammation linked to the expression of the remaining viral genes. They can only be propagated by transcomplementation of the adenovirus functions for which they are deficient. Currently, one uses essentially the complementation line 293 (Graham et al., 1977, J. Gen. Virol. 36, 59-72) or lines derived therefrom (Yeh et al., 1996, J. Virol. 70, 559-565; Wang et al., 1995, Gene Therapy 2, 775-783; Krougliak and Graham, 1995, Human Gene Therapy 6, 1575-1586).
In particular, adenoviruses are used for treating cystic fibrosis by gene therapy (Pavirani et al., 1996, medecine/sciences 12, 25-33).
However, recombinant viruses can only be used if their viability and their infectiveness have been adequately preserved over the entire storage period.
Purified adenoviruses are traditionally preserved in saline containing 10 to 30% of glycerol (Graham et al., 1991, Methods in Molecular Biology, vol. 7, chapter 11, p. 109-127; Ed Murrey, The Human Press Inc.; Precious and Russel, Virology, a Practical Approach, 1985, chapter 9, p. 193-205; ed: BW Mahy, IRL Press, Washington DC; Kanegae et al., Jpn. J. Med. Sci. Biol., 47, 157-166, 1994 and Green et al., Methods in enzymology, vol. LVIII, p. 425-435). However, the glycerol has the disadvantage of irritating the pulmonary epithelium, which may be tricky in the case of intratracheal and intrapulmonary administration (for example for the treatment of cystic fibrosis or of cancers of the pulmonary tract). In addition, while this solution allows adenoviruses to be preserved in frozen form, it does not allow their activity to be maintained at +4xc2x0 C. beyond one week.
Addition of sucrose at a low concentration (1 to 5%) to a saline has also been described (Precious et al., see above; Huyghe et al., Human Gene Therapy 6: 1403-1416, November 1995, and Rehir, Process Development and Production Issues for Viral Vectors and Vaccines, The Williamsburg Bio Processing Conference, 2nd annual meeting, Nov. 6-9, 1995), which allows long-term stability of the adenoviruses in frozen form, but at +4xc2x0 C. only in the short term (Hehir, see above).
Since preservation of viruses in frozen form presents storage and transport problems, it has also been envisaged to preserve the viruses and the viral vaccines in lyophilized form. However, this technique has the disadvantage that it frequently entails loss of viral activity. To make up for this, addition of excipients such as sugars (sucrose, glucose, trehalose) allows the viral activity to be maintained in lyophilized form (WO 95/10601xe2x80x94Viagene and EP-0 357 709xe2x80x94Quadrant). The use of lactose or sucrose at low concentrations (2.5-5%) for the preservation of attenuated live viruses in lyophilized form has also been recommended (JP-88 555465xe2x80x94Kitasako Inst.).
None of the solutions proposed to date has permitted maintaining the activity of adenoviruses at satisfactory levels over more than 6 months while avoiding secondary problems such as problems with irritation.
The present invention overcomes the shortcomings of the prior art. It relates to a long-term preservation method for infectious recombinant viruses, both in liquid form and in frozen form, in which the recombinant viruses are preserved in an aqueous solution which comprises sucrose at high concentration.
In effect, even though the use of sucrose at high concentration has been known for a long time for the preservation of proteins or other biological products (Timasheef et al., In Protein Structure, a Practical Approach, 1989, Ed Creighton, chapter 14, p.331-345, IRL Press, Oxford, and Doebbler, Cryobiology, vol. 3, No. 1, 1966) or for the cryopreservation of live cells in liquid nitrogen (Grout et al., Tibtech, October 1990, vol. 8, p. 293-297), it has never been proposed for the reservation of viruses.
The results obtained by making use of the process according to the invention have now demonstrated a cryoprotective effect of sucrose at different storage temperatures (xe2x88x9280xc2x0 C., xe2x88x9240xc2x0 C., xe2x88x9220xc2x0 C. and +4xc2x0 C.) and this is more pronounced the higher the sucrose concentration.
The infectious recombinant viruses to which the present invention relates are advantageously poxviruses, adenoviruses, viruses associated with adenoviruses and retroviruses.
Within the frame of the invention, the viruses are preserved in an aqueous solution comprising sucrose at high concentration, that is to say at a concentration of above 0.75 M, preferably between 0.75 M and 1.5 M, more preferably at a concentration of 1 M.
In accordance of an advantageous embodiment of the method according to the invention, the infectious recombinant viruses gain in stability when the aqueous solution used has a basic pH of between 8 and 9, preferably 8.5.
Thus, the aqueous solution of which use is made within the frame of the present invention can be a buffer solution selected from amongst Tris buffer, and triethanolamine, diethanolamine, borate/HCl, glycine/NaOH, EPPS [N-(2-hydroxyethyl)piperazine-Nxe2x80x2-(3-propanesulfonic), acid], bicine, TAPS [N-Tris-(hydroxymethyl)-methyl-3-aminopropanesulfonic acid] and tricine solutions.
Advantageously, it is furthermore possible to stabilize the capsid or viral coat of the viruses preserved according to the invention by adding, to the aqueous solution used, at least one salt of a divalent cation selected from amongst MgCl2, CaCl2 and MnCl2, with MgCl2 being preferred.
Within the frame of the present invention, the salt of the divalent cation is used at a concentration of between 0.1 and 5 mM, preferably between 0.5 and 2 mM, ore preferably in the order of 1 mM.
According to an advantageous embodiment of the method according to the invention, the viruses are preserved in a buffer solution comprising 10 mM Tris-HCl buffer, 1 mM MgCl2, 1 M sucrose, pH 8.5.
Preservation of the viruses can be improved even further by using at least one stabilizer selected from amongst salts, preferably monovalent salts such as NaCl or KCl, which impart an ionic strength to the solution, amino acids such as Gly, Leu, Lys, Arg, Asp, Val, Glu and compounds which act on the surface tension such as TWEEN 80 (polysorbate 80) or TRITON X-100, (nonaethylene glycol octyl phenol ether), it being possible to use the latter items alone or in the presence of salts.
By way of stabilizer, the NaCl is advantageously used at a concentration of between 0.05 and 1 M, preferably between 0.1 and 0.5 M, more preferably between 0.1 and 0.3 M, the concentration considered as optimum being 0.15 M, and the TWEEN 80 is used at a concentration between 0.001 and 0.5% by weight based on the total solution (that is to say between 10 mg/i and 5 g/l), preferably between 0.002 and 0.2% by weight, more preferably in the order of 0.005% by weight.
According to a preferred embodiment, the method according to the invention makes use of an aqueous solution of a pH of approximately 8.5 comprising 10 mM Tris-HCl, 1 MM MgCl2, 0.9% (or 150 mM) NaCl, 50 mg/l (0.05%) TWEEN 80 and 1M sucrose 1.
In addition, the infectious recombinant viruses preserved in accordance with the method according to the invention may be lyophilized.
The invention furthermore relates to an aqueous suspension of infectious recombinant viruses in an aqueous sucrose solution at high concentration as described above.
The aqueous suspension according to the invention advantageously comprise 106 to 1013 pfu/ml infectious recombinant virus.
The present invention also relates to a pharmaceutical composition comprising an aqueous suspension of infectious recombinant viruses such as described above or obtained by making use of the preservation method according to the invention, in association with a pharmaceutically acceptable vehicle. It can be administered by the systemic route, in particular the subcutaneous, intravenous, intracardiac, intramuscular, intraperitoneal, intragastric, intratumoral, intrapulmonary, intranasal or intratracheal route. Administration can be as a single dose or a dose which is repeated once or more than once after a certain interval. Also, the formulation may include other compounds such as an adjuvant or pharmaceutically acceptable excipient. In particular, a composition according to the invention is intended for the preventative or curative treatment of diseases such as genetic diseases (hemophilia, cystic fibrosis, diabetes, Duchenne""s and Becker""s myopathy, . . . ), cancers, viral diseases (various forms of hepatitis, AIDS, . . . ) and recurrent diseases (infections provoked by herpesvirus, human papilloma virus, . . . ).